Wang et al. Stem Cell Research & Therapy (2017) 8:245 DOI 10.1186/s13287-017-0698-8

RESEARCH Open Access

Using low-risk factors to generate non-integrated human induced pluripotentstem cells from urine-derived cells

Linli Wang1*†, Yuehua Chen1†, Chunyan Guan1†, Zhiju Zhao1, Qiang Li1, Jianguo Yang1, Jian Mo1, Bin Wang1,Wei Wu2, Xiaohui Yang1, Libing Song3 and Jun Li1,4*

Abstract

Background: Because the lack of an induced pluripotent stem cell (iPSC) induction system with optimal safety andefficiency limits the application of these cells, development of such a system is important.

Methods: To create such an induction system, we screened a variety of reprogrammed plasmid combinations andmultiple compounds and then verified the system’s feasibility using urine cells from different individuals. We alsocompared large-scale iPSC chromosomal variations and expression of genes associated with genomic stabilitybetween this system and the traditional episomal system using karyotype and quantitative reverse transcriptionpolymerase chain reaction analyses.

Results: We developed a high-efficiency episomal system, the 6F/BM1-4C system, lacking tumorigenic factors forhuman urine-derived cell (hUC) reprogramming. This system includes six low-risk factors (6F), Oct4, Glis1, Klf4,Sox2, L-Myc, and the miR-302 cluster. Transfected hUCs were treated with four compounds (4C), inhibitor oflysine-demethylase1, methyl ethyl ketone, glycogen synthase kinase 3 beta, and histone deacetylase, within ashort time period. Comparative analysis revealed significantly decreased chromosomal variation in iPSCs andsignificantly increased Sirt1 expression compared with iPSCs induced using the traditional episomal system.

Conclusion: The 6F/BM1-4C system effectively induces reprogramming of urine cells in samples obtained fromdifferent individuals. iPSCs induced using the 6F/BM1-4C system are more stable at the cytogenetic level andhave potential value for clinical application.

Keywords: Induced pluripotent stem cells, Human urinary cells, 6F/BM1-4C system, iPSC safety

BackgroundAdvancements in induced pluripotent stem cell (iPSC)technology have provided great opportunities for regenera-tive medicine and tumor immunotherapy [1–3]. However,human iPSCs (hiPSCs) are primarily induced using retro-viral or lentiviral vectors carrying reprogramming factors[4, 5], and exogenous DNA fragments can randomly insertinto genomic DNA and induce cell transformation, therebypreventing the clinical application of iPSCs. To date, manynon-integrating methods have been generated, including

* Correspondence: linli_wang@163.com; lijun37@mail.sysu.edu.cn†Equal contributors1Guangzhou Biocare Institute of Cancer, Building D, Guangzhou InternationalBusiness Incubator, No. 3, Juquan Road, Guangzhou Science Park,Guangzhou 510663, Guangdong, People’s Republic of ChinaFull list of author information is available at the end of the article

© The Author(s). 2017 Open Access This articInternational License (http://creativecommonsreproduction in any medium, provided you gthe Creative Commons license, and indicate if(http://creativecommons.org/publicdomain/ze

mRNA and protein transfection [6, 7], Sendai virus (SeV)[8], piggyback (PB) transposons [9], and episomal vectors[10]. Nonetheless, mRNA and protein transfections areassociated with high preparation costs and low inductionefficiency, and a risk of transformation is associated withthe retention of SeV RNA in the first passages of iPSC lines[8, 11] and PB transposons. Although episomal inductionsystems can avoid these problems and are widely used forreprogramming, most of these systems utilize at leastone tumorigenic factor such as c-Myc, SV40-LT, andp53 inhibitors, including p53 RNA interference (RNAi)or small molecule inhibitors [10, 12–17]. As inductionefficiency varies when the same method is used toreprogram different types of somatic cells or when differentmethods are applied to reprogram the same type of somatic

le is distributed under the terms of the Creative Commons Attribution 4.0.org/licenses/by/4.0/), which permits unrestricted use, distribution, andive appropriate credit to the original author(s) and the source, provide a link tochanges were made. The Creative Commons Public Domain Dedication waiverro/1.0/) applies to the data made available in this article, unless otherwise stated.

Wang et al. Stem Cell Research & Therapy (2017) 8:245 Page 2 of 13

cells [12], it is crucial to optimize the induction method foreach type of cell. Human urine-derived cells (hUCs) areideal donors for iPSC generation: their isolation is simpleand nontraumatic. In addition, these cells are easy to ex-pand in vitro and can be used as the main source for iPSCs;thus, their use is cost-effective and universal [18–20]. In thepresent study, an episomal vector was used for hUC repro-gramming [14, 17].The nontransformative MYC family protein L-Myc can

be replaced with c-Myc to induce iPSCs [15, 21]. Glis1,which is enriched in oocytes, can also replace c-Myc inthe classical OSKM (Oct4, Sox2, Klf4, and c-Myc) system;chimeric mice generated from iPSCs induced using OSKand Glis1 have longer survival times than those generatedfrom iPSCs induced by OSKM [22]. Moreover, there is apositive correlation between chimeric mouse mortality andmouse tumor mortality [21], suggesting that Glis1 is asafety factor for iPSC generation. The miR-302 family,which is specifically expressed in embryonic stem cells(ESCs), can partially or completely replace reprogram-ming factors and increase reprogramming efficiency[14, 23, 24]. Furthermore, the miR-302 family activatesInk4a and Arf to suppress the tumorigenesis of humanpluripotency stem cells by targeting the oncogene Bmi1[25], and Arf/p53 pathway activation suppresses somaticcell reprogramming [15, 16]. Therefore, miR-302 s are typ-ically important factors that promote somatic cell repro-gramming, but targeted factors that inhibit reprogrammingexist in some signal pathways. Several studies to datehave suggested that long noncoding RNAs (lncRNAs)regulate development and tumorigenesis; for example,long intergenic noncoding RNA, regulator of reprogram-ming (lincRNA-ROR) regulates the self-renewal and pluri-potency of human ESCs (hESCs) and the reprogrammingof hiPSCs [26, 27]. In this study, we applied hUCs asdonor cells to induce iPSCs using low-risk factors, andthen we screened a combination of low-risk reprogram-ming factors, including Oct4, Glis1, Klf4, Sox2, L-Myc, andthe miR-302 cluster.To improve non-integrated reprogramming efficiency,

we optimized our culture system and observed iPSC induc-tion with high efficiency when four compounds (Parnate,PD0325901, CHIR99021, and sodium butyrate) were addedto the medium for no more than 4 days. To analyze iPSCsafety, a karyotype analysis was performed, and theresults showed that significantly lower iPSC chromo-somal variation was induced when using this systemthan when using episomal systems containing SV40-LTand c-Myc.

MethodsCell culturehUCs were collected according to methods reported previ-ously [14, 18]. Briefly, 100–1000 ml of urine was collected

from donors, centrifuged at 1010 × g for 5 minutes, andwashed with phosphate-buffered saline (PBS). The cellswere maintained in 24-well plates coated with 0.1% gel-atin (ES-006-B; Millipore, Germany) in RM1 medium(50% Renal Epithelial Cell Growth Medium (REGM)(CC-3190; Lonza, USA) and 44% Dulbecco's ModifiedEagle Medium (DMEM) (SH30022; HyClone, USA)supplemented with 5% fetal bovine serum (FBS) (P30-3302; PAN Biotech, Germany), 0.5% nonessential aminoacids (NEAA) (11140050; Gibco, USA), 0.5% GlutaMax(35050-061; Gibco, USA)) and 1 × Primocin (ant-pm-2;InvivoGen, USA); 0.25% trypsin-EDTA (25200072; Gibco,USA) was used for dissociation of primary hUCs. RM1 orRM2 (82% DMEM (SH30022; HyClone, USA) supple-mented with 5% FBS, 1% human keratinocyte growth sup-plement (HKGS) (S-001-5; Gibco, USA), 1% NEAA, and1% GlutaMax) was used for hUC culture.The HN4 hESC line was obtained from the Chinese

Academy of Sciences, and both HN4 and hiPSCs weremaintained in the hESC medium BioCISO (BC-PM0001;BIOCARE Biotech, China) in plates coated with Matrigel(354277; Corning, USA).

PlasmidspCEP4 (V04450; Invitrogen, USA) was digested usingthe restriction enzymes NruI and SalI and ligated withsynthesized multiple cloning site (MCS) oligonucleotidesto obtain the plasmid pE2.1. The EF1α promoter (NruI,NheI), BGH-PA element (BamHI, PmeI), EF1α promoter(PmeI, BglII), and BGH-PA element (PacI, SalI) werecloned into pE2.1 to obtain the plasmid pE3.1. Theprocess chart for pE3.1 plasmid construction is shown inAdditional file 1: Figure S1a. To obtain the plasmidpE3.2, the EF1α promoter (Pme I, Bgl II) was replacedwith the cytomegalovirus (CMV) promoter (PmeI, BglII).The sequences of Oct4, Sox2, and Klf4 were subclonedfrom OKSIM (Plasmid 24603; Addgene, USA). The Glis1sequence was subcloned from pMXs-Glis1 (Plasmid 30166;Addgene, USA) and the L-Myc sequence from pMXs-Hu-L-Myc (Plasmid 30166; Addgene, USA). The Oct4-P2A-Glis1, Klf4-P2A-Sox2, and Oct4-P2A-L-Myc sequenceswere obtained using overlap PCR. The sequence of themiR-302 cluster was cloned from genomic DNA. The exonsequences of lincRNA-ROR were amplified using genomicDNA and synthesized DNA, and overlap PCR was used toobtain the complete lincRNA-ROR sequence. These DNAsequences were cloned into the plasmids pE3.1 and pE3.2,respectively, to generate the plasmids pE3.1-OL--KS,pE3.1-OG--KS, pE3.1-Oct4--Klf4, pE3.1-Glis1--LINC-ROR,pE3.1-L-Myc--hmiR-302 cluster, pE3.1-Oct4--Sox2, andpE3.2-L-Myc--hmiR-302 cluster. Information regardingthe factors, primer sequences, and MCS is shown inAdditional file 2: Table S1.

Wang et al. Stem Cell Research & Therapy (2017) 8:245 Page 3 of 13

iPSC generationFor hUC16 reprogramming, 1.5 × 106 hUC16 cells weretransfected with plasmids (Additional file 3: Table S2)using the T-020 program of a Lonza Nucleofector 2bDevice and a Basic Epithelial Cells Nucleofector Kit(VPI-1005; Lonza, USA). Transfected hUC16 cells wereseeded into six-well plates coated with 0.1% gelatin andcultured using RM2 medium. After 24 h, the cells weredissociated using 0.25% trypsin-EDTA (25200-072; Gibco,USA), and 2 × 104 cells were seeded into 12-well platescoated with Matrigel. To induce iPSCs, the medium waschanged to BioCISO-BM1 medium (BC-BM001; BIOCAREBiotech, China) containing 4i (A83-01 (0.5 μM, BC-SMC-A01-10; BIOCARE Biotech, China), Thiazovivin (0.5 μM,BC-SMC-T01-10; BIOCARE Biotech, China), CHIR99021(3 μM, BC-SMC-C01-10; BIOCARE Biotech, China),and PD03254901 (0.5 μM, BC-SMC-P01-10; BIOCAREBiotech, China)) after 24 h. The medium was then changedto BioCISO on day 15. Alkaline phosphatase (AP) stainingwas performed on day 18, and the induction efficiency wascalculated according to the formula:

Induction efficiency ¼ AP‐positive colony number=total seeded cell number � 100%:

For reprogramming using the 6F/BM1-4C system, ap-proximately 2.8 × 106–3.5 × 106 hUCs were transfectedwith 4.0 μg pE3.1-OG--KS and 2.8 μg pE3.1-L-Myc--hmiR-302 cluster using the same nucleofector method.The transfected hUCs were placed in plates coated withMatrigel and cultured with RM1 medium. On day 3after nucleofector addition, the medium was changedto BioCISO-BM1 medium containing 2 μM Parnate(also known as tranylcypromine hydrochloride, 1986-47-6;Curegenix, China). The medium was then changed toBioCISO-BM1 medium containing 2 μM Parnate, 0.25 mMsodium butyrate (NaB) (303410-100G; Sigma, USA), 3 μMCHIR99021, and 0.5 μM PD03254901 on day 5, toBioCISO-BM1 on day 7, and to BioCISO on day 17.iPSC colonies were collected or stained with AP on day19. The induction efficiency was calculated accordingto the formula:

Induction efficiency ¼ AP‐positive colony number=ðnucleofector cell number– death cell numberÞ � 100%:

Compounds used in the present study also includeddimethyloxaloylglycine (DMOG) (0.1 μM, D1070; FrontierScientific, USA), PS48 (5 μM, 1180676-32-7; Curegenix,China), SC-79 (0.5 μM, 4635; Tocris, USA), forskolin (5 μM,66575-29-9; Curegenix, China), and 3-deazaneplanocin A(DZNEP) (0.05 μM, 4703; Tocris, USA).For reprogramming using the 4F2L-6C system, 3.0 × 106

hUCs were transfected with 4.0 μg pEP4-E02S-ET2K and2.8 μg pCEP4-M2L using the same nucleofector method.

The transfected hUCs were placed in plates coated withMatrigel and cultured with RM1 medium. The mediumwas changed to BioCISO-BM1 medium containing 2 μMParnate on day 3 after nucleofector addition, and thenchanged to BioCISO-BM1 medium containing 2 μMParnate, 0.25 mM NaB, 3 μM CHIR99021, 0.5 μMPD03254901, 0.5 μM A83-01, and 0.5 μM Thiazovivin onday 5. The medium was changed to BioCISO-BM1 on day7 and to BioCISO on day 17. iPSC colonies were collectedon day 34.

iPSC characterizationAP staining, non-integrated PCR analysis, flow cytome-try analysis, immunofluorescence analysis, bisulfate se-quencing, in-vitro embryoid body (EB) differentiationassays, and in-vivo teratoma formation were conductedaccording to previous methods [14, 17]. Briefly, AP stain-ing was carried out using nitroblue tetrazolium (NBT)(N104908-1 g; Aladdin, China) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (BIMB1018; J&K ChemicalTechnology, China). PCR was applied to analyze the in-tegration of exogenous reprogramming factors and the epi-somal backbone with the primers presented in Additionalfile 2: Table S1. Flow cytometry and immunofluorescenceanalyses were employed to examine human pluripotencymarkers with the following antibodies: anti-Oct4 (130-105-606; Miltenyi Biotec, Germany), anti-SSEA4 (sc-21704;Santa Cruz, USA), anti-Tra-1-60 (sc-21705; Santa Cruz,USA), anti-Tra-1-81 (sc-21706; Santa Cruz, USA), anti-IgM-PE (sc-3768; Santa Cruz, USA), and anti-IgG3-PE(sc-3767; Santa Cruz, USA). Bisulfate sequencing wasused to determine Oct4 and Nanog promoter methylationwith the primers presented in Additional file 2: Table S1.The PCR product was cloned into the pMD18-T vectorand subsequently sequenced. For the in-vitro EB differenti-ation assay, cells were scraped from plates after dissociationusing BioC-PDE1 (BC-PDE1; BIOCARE Biotech, China)and cultured in six-well suspension culture plates (657185;Greiner, Germany) with BioCISO-EB1 medium (BC-EB001;BIOCARE Biotech, China) for 7 days to obtain EBs. TheEBs were then cultured in six-well culture plates (657160;Greiner, Germany) coated with Matrigel for 7–14 days.The cells were collected, and expression profiles ofmarker genes in the three germ layers were determinedusing quantitative reverse transcription PCR (qRT-PCR)with primers obtained from BIOCARE Biotech (BioEB-pri-mer). For in-vivo teratoma formation, iPSCs were culturedto approximately 85% confluence; after 10–15 minutes ofdissociation using BioC-PDE1, cells were scraped from theplates. The hind-limb muscle and forelimb subcutaneousmuscle of 6-week-old NOD/SCID mice were injected with130 μl BioCISO culture medium, 70 μl Matrigel, and cellsuspensions. The formation of teratomas could be observedafter 6–8 weeks; when the teratomas reached a certain size,

Wang et al. Stem Cell Research & Therapy (2017) 8:245 Page 4 of 13

they were removed and fixed with 4% paraformaldehyde.The tissues were embedded with paraffin, sectioned,stained with hematoxylin and eosin, and analyzed under amicroscope. The procedures were performed according toIACUC (Institutional Animal Care and Use Committee;YS-YFStudy060-20160315).

Karyotype analysisSample preparation for karyotyping was conducted asdescribed previously [28]. Cells were treated with 50 ng/mlcolchicine (Xy008; Xiangya Gene Technology, China)for 16 h, and the Ikaros karyotyping system was used toanalyze karyotypes. The aneuploid evaluation is shownin Additional file 4: Table S3.

Quantitative reverse transcription polymerase chain reactionTotal RNA was isolated using RNAiso Plus (TaKaRa),and M-MLV Reverse Transcriptase (TaKaRa) was usedto synthetize cDNA. Specific stem-loop primers and ran-dom primers were used for reverse transcription of micro-RNAs and mRNAs into cDNA, respectively. mRNA andmiRNA expression levels were determined using SYBRPremix Ex Taq™ (TaKaRa). Reactions were performed intriplicate using a LightCycler 480II/96 system (Roche,Switzerland). mRNA expression was normalized to GAPDH,and microRNA (miRNA) expression was normalized to U6small nuclear RNA (snRNA). The primers are presented inAdditional file 2: Table S1.

Western blot analysisRadioimmunoprecipitation assay (RIPA) buffer (CW2333;Cwbiotech, China) supplemented with a protease inhibitorcocktail (PI003; BOCAI Technology, China) and phenyl-methylsulfonyl fluoride (PMSF; Dingguo ChangshenBiotech, China) was used to isolate cellular proteins.Equivalent amounts of protein were separated by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF)membranes. The membranes were incubated with specificprimary antibodies against Oct4 (2840; Cell SignalingTechnology, USA), Glis1 (SAB2700289; Sigma, USA), Klf4(ab72543; Abcam, UK), Sox2 (3579; Cell Signaling Technol-ogy, USA), L-Myc (sc-790; Santa Cruz Biotechnology, USA),and GAPDH (KC-5G4; KangChen Biotech, China), followedby horseradish peroxidase-conjugated secondary antibodies:goat anti-Rabbit IgG (ZB-2301; ZsBio, China) and anti-mouse IgG-HRP (IH-0031; Dingguo Changshen Biotech,China). Bands were visualized using enhanced chemilu-minescence (ECL) (34087; Thermo, USA).

Microarray analysisGeneChip Human Transcriptome Array 2.0 (AffymetrixHTA 2.0, USA) was utilized to determine the gene ex-pression profiles of human ESCs, iPSCs, and hUCs. The

experiments were conducted according to the manufac-turer’s instructions.

Statistical analysisSPSS 18.0 was used to perform statistical analysis. Theresults are presented as the mean ± standard deviation (SD)of at least three repeated individual experiments for eachgroup. Statistical differences were examined using Student’st test. For analysis of the chromosome abnormality rate, afour-table chi-square test was applied. P < 0.05 was consid-ered statistically significant.

Accession numbersMicroarray data for human ESs, iPSCs, and hUCs havebeen submitted to Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession numberGSE85885.

ResultsScreening low-risk reprogramming factors using hUCsSince Yamanaka used OSKM to induce reprogramming,many genes and non-RNAs that improve reprogrammingefficiency have been reported. To induce reprogramming inthis study, we employed low-risk factors, including L-Myc,Glis1, lincRNA-ROR, and the miR-302 cluster, and ran-domly combined them with Oct4, Sox2, and Klf4 in theEpstein–Barr virus-encoded nuclear antigen-1 (EBNA)-oriPepisomal vector (Additional file 1: Figure S1a, S1b, S1c).The miR-302 family can increase reprogramming efficiencyby replacing reprogramming factors [14, 23, 24]. Further-more, miR-302 family members target the oncogene Bmi1and suppress tumorigenesis, which can inhibit somatic cellreprogramming [15, 16, 25]. Therefore, miR-302 s havea positive impact on somatic cell reprogramming, butin some pathways these family members can inhibitreprogramming by indirectly activating targets that inhibitreprogramming. Because the expression levels of the miR-302 cluster must be precisely regulated, we used differentpromoters to exogenously express the miR-302 clusterand screened for optimal expression (Additional file 1:Figure S1a, S1b).To evaluate the best plasmid combination for reprogram-

ming, we used hUC16 cells constructed in our laboratorythat showed high proliferation (Additional file 5: FigureS2a) and transfected these cells with different plasmidcombinations (Additional file 5: Figure S2b), followedby AP staining to identify iPSCs after 18 days of nucleofec-tion. Three groups of cells harboring the reprogrammingfactors Oct4, Glis1, Klf4, Sox2, L-Myc, lincRNA-ROR, andthe miR-302 cluster with high AP-positive scores were se-lected for further analysis (Additional file 5: Figure S2c,S2d). The initial screen was performed in cells (UC16) withhigh proliferative ability and strong anti-stress capacity, andthe best three combinations were further tested using other

Wang et al. Stem Cell Research & Therapy (2017) 8:245 Page 5 of 13

hUCs to ensure reliability. To induce reprogramming, wetransfected the three plasmid combinations into hUCs andcultured in hESC basal medium (BioCISO-BM1) with theaddition of four small inhibitors that have been widely usedfor reprogramming [12, 29]: 4i, the TGF-β/Activin/Nodalreceptor inhibitor A83-01 (0.5 μM); the MEK inhibitorPD0325901 (0.5 μM); the GSK3β inhibitor CHIR99021(3 μM); and the ROCK inhibitor Thiazovivin (0.5 μM)(Fig. 1a, b). As revealed by AP staining, the combinationtermed 6F, which includes Oct4, Glis1, Klf4, Sox2, L-Myc,and the miR-302 cluster initially expressed from the CMVpromoter (Fig. 1c, d), showed the highest reprogrammingefficiency at 19 days post nucleofection. Thus, we selected6F, which does not contain high-risk tumorigenic factorssuch as c-Myc, SV40-LT, and p53 inhibitors, as the repro-gramming induction combination for hUCs and found thatit successfully induced hUCs into iPSCs.

0

1

2

3

4

5

6

7

8

9

10

A B C

Num

ber

Of A

P+ C

loni

es/5

X10

4 C

ells * *

C

B

A

Trypsinization

D0 D1 D2 D5 D16 D19

RM1RM1 RM1+3i BioCISO-BM1+4i BioCISO

Electroporation AP

_++

+ _ +_ + _

_ +_

_ +_pE3.1-OG--KS

PlasmidGroup

pE3.2-L-Myc-hmiR-302cluster

pE3.1-OL--KS

B CA

pE3.1-L-Myc-hmiR-302cluster

pE3.1-Glis1--LINC-ROR

a

b

c d

Fig. 1 Screening low-risk factors for iPSC generation. a Screening strategyfor the optimal plasmid combination. b Time schedule of iPSC generation.3i = 0.5 μM A83-01, 3 μM CHIR99021, 0.5 μM thiazovivin. c AP staining toidentify iPSCs. d Number of AP-positive colonies. Error bars indicate mean± SD. *P< 0.05. P(B) = 0.038, P(C) = 0.022. AP alkaline phosphatase, D day

Optimization of compounds in the 6F combination systemDifferent cell lineages exhibit different gene expressionprofiles, and ideal reprogramming factor combinations andinduction conditions depend on the cell type [12, 30, 31].To determine whether 4i is the best induction condition forthe 6F combination, we first examined the effects of com-pounds that are reported to regulate reprogramming in oursystem, including many compounds involved in signalingpathways. Some inhibitors or activators were observed tobe unsuitable for our system; for example, the TGF-β/Acti-vin/Nodal receptor inhibitor A83-01 and the ROCK inhibi-tor Thiazovivin did not promote programming (Additionalfile 6: Figure S3a–S3f). In addition, other compounds[31, 32], such as the PDK1 activator PS48, adenylyl cy-clase activator forskolin, histone methyltransferase in-hibitor DZNEP, HIF-α prolyl hydroxylase inhibitor DMOG,and Akt activator SC-79, did not promote or inhibit pro-gramming (Additional file 6: Figure S3g–S3l). Next, weexamined the effects of combinations of compounds thatpromote reprogramming on the reprogramming efficiency.These compounds included the MEK inhibitor PD0325901(PD, 0.5 μM), the GSK3β inhibitor CHIR99021 (CHIR,3 μM), the histone deacetylase inhibitor NaB (0.25 mM),and the lysine-specific demethylase 1 inhibitor Parnate (Par,2 μM). We observed that when Par was used in 6F combin-ation reprogramming for an extended time interval, manycells in the cell layer shrank and were floating; however, thereprogramming efficiency increased when Par was addedfor a short time period (Additional file 6: Figure S3m–S3q).Thus, compound combinations, including Par, were usedfor no longer than 4 days. AP staining showed the high-est reprogramming efficiency and the best iPSC qualitywhen Par was added to the BioCISO-BM1 medium ondays 3–4 and when PD, CHIR, NaB, and Par, termed4C, were added on days 5–6 (Fig. 2a–c). Therefore, weselected BM1-4C as the optimum induction culturecondition for the 6F combination induction system andnamed it 6F/BM1-4C.

Reprogramming hUCs from different human sourcesusing the 6F/BM1-4C systemWhen hUCs were isolated from different individuals orfrom the same individual at different time points, thecells exhibited different morphologies when cultured,suggesting that these cells were of different types, whichis consistent with previous reports [17, 33–35]. To confirmthat the 6F/BM1-4C system is suitable for reprogrammingdifferent types of hUCs, we reprogrammed seven groups ofhUCs isolated from different individuals (Fig. 3a, b, Table 1).At approximately 19 days post nucleofection, we col-lected iPSCs for further purification and expansion culture(Fig. 3c). The remaining iPSCs were subjected to AP stain-ing, which demonstrated that the 6F/BM1-4C system re-sulted in effective reprogramming of all hUCs into iPSCs.

a

bA B C D E F

LKJIHG

c

A

B

C

D

E

F

G

H

I

J

K

L

RM1

D0-D2 D3-D4 D7-D16 D17-D19D5-D6

RM1

RM1+Par

RM1+Par

RM1+Par

RM1+Par

RM1+Par

BioCISO-BM1+Par

BioCISO-BM1+Par

BioCISO-BM1+Par

BioCISO-BM1+Par

BioCISO-BM1+Par

BioCISO-BM1

BioCISO-BM1

BioCISO-BM1+Par

BioCISO-BM1+Par+NaB

BioCISO-BM1+Par+NaB+CHIR

BioCISO-BM1+Par+NaB+PD

BioCISO-BM1+Par+NaB+CHIR+PD

BioCISO-BM1+Par

BioCISO-BM1+Par+NaB

BioCISO-BM1+Par+NaB+CHIR

BioCISO-BM1+Par+NaB+PD

BioCISO-BM1+Par+NaB+CHIR+PD

BioCISO-BM1BioCISO-BM1 BioCISO

Group Time

0

5

10

15

20

A B C D E F G H I J K L

N

um

ber

Of A

P+

Clo

nie

s / 3X

10

4 C

ells

*

***

**

********

Fig. 2 Optimization of the 6F combination system. a Screening strategy for six-factor combinations. b AP staining to evaluate iPSC generation.c Numbers of AP-positive iPSCs induced using different strategies. Error bars indicate mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. P(B) = 0.038,P(C) = 0.022, P(D) = 0.006, P(I) = 0.002, P(J) = 0.001, P(K) = 0.000, P(L) = 0.000. AP alkaline phosphatase, D day, PD PD0325901, CHIR CHIR99021, NaBsodium butyrate, Par Parnate

Wang et al. Stem Cell Research & Therapy (2017) 8:245 Page 6 of 13

AP staining also revealed an AP-positive rate that variedbetween 0.00021 and 0.0741% for iPSCs reprogrammedfrom different types of hUCs (Fig. 3d, Table 1). Further-more, PCR analysis demonstrated a lack of genomic inte-gration of the exogenous gene sequence in 13 of 14 iPSCcolonies (Fig. 3e, Additional file 7: Figure S4a). Moreover,karyotype analysis revealed that 13 iPSC colonies hadnormal chromosome numbers and G-band distributions(Fig. 4a, Additional file 7: Figure S4b). Flow cytometryexpression and immunofluorescence analyses revealed thatiPSCs induced using the 6F/BM1-4C system express thehESC-specific markers Oct4, SSEA4, Tra-1-60, and Tra-1-81, suggesting that 6F/BM1-4C-iPSCs possess the molecu-lar characteristics of hESCs (Fig. 4b, c, Additional file 8:Figure S5a, S5b). In addition, bisulfite sequencing PCR ana-lysis indicated that the endogenous pluripotency genesOct4 and Nanog were activated and that their promoterswere demethylated in 6F/BM1-4C-iPSCs, similar towhat occurs in hESCs (Fig. 4d, Additional file 8: Figure S5c).

The generated iPSCs were also able to differentiate into de-rivatives of all three germ layers, as determined usingan in-vitro EB differentiation assay and an in-vivo tera-toma formation assay (Fig. 4e, f, Additional file 8: FigureS5d, S5e). The gene expression profile of iPSCs was simi-lar to that of hESCs and differed from that of hUCs, whichwas determined using Affymetrix gene microarray HTA2.0. (Fig. 4g, Additional file 8: Figure S5f). The inductionefficiencies of the different sources of hUCs as well as theiPSC characteristics are presented in Table 1. Together,these results indicate that the 6F/BM1-4C system has highreliability and versatility for reprogramming hUCs intoiPSCs.

The 6F/BM1-4C system is safer than traditional episomalinduction systemsWe observed that before day 5 following nucleofection,the observed cell masses had high nuclear–cytoplasmicratios, a phenotype that was similar to the lentiviral

UC1 UC2 UC3 UC4 UC5 UC6 UC7

UC1 UC2

UC3 UC4

UC5 UC7

d e1 2 3 4 5 6 7 8 9 101112 131415161718

OriP

Oct4 endo

L-Myc

Sox2

Klf4Glis1

EBNA1

(1)

(2)

(3)

Oct4

302cluster

(1)

(2)

(3)

(1)

(2)

(1)

(2)

(1)

(2)

(3)

b

a

D0 D3 D5 D7 D17

RM1BioCISO-BM1+Par

BioCISO-BM1+4C

BioCISO-BM1 BioCISO

AP

D19

cUC2iPSC1 UC3PSC1 UC4iPSC1 UC7iPSC1UC5iPSC1 UC6iPSC2UC1iPSC1

Electroporation

Fig. 3 Induction of iPSCs from multiple hUCs using the 6F/BM1-4C system. a Morphology of hUCs isolated from seven different donors. b Timeschedule of the 6F/BM1-4C reprogramming system. c Morphology of iPSCs induced from seven groups of hUCs using the 6F/BM1-4C system. dAP staining for iPSC generation from multiple hUCs using the 6F/BM1-4C system. e Non-integrating analysis of episomal DNA in iPSCs. Representativelanes: 1, H2O; 2, pE3.1-OG--KS and pE3.2-L-Myc--hmiR-302 cluster; 3, UC1; 4, UC1, pE3.1-OG--KS, and pE3.2-L-Myc- -hmiR-302 cluster; 5, UC1iPSC1; 6,UC1iPSC2; 7, UC2; 8, UC2, pE3.1-OG--KS, and pE3.2-L-Myc- -hmiR-302 cluster; 9, UC2iPSC1; 10, UC2iPSC2; 11, UC3; 12, UC3, pE3.1-OG--KS, andpE3.2-L-Myc- -hmiR-302 cluster; 13, UC3iPSC1; 14, UC3iPSC2; 15, UC4; 16, UC4, pE3.1-OG--KS, and pE3.2-L-Myc- -hmiR-302 cluster; 17, UC4iPSC1; 18,UC4iPSC2. Scale bars, 100 μm. AP alkaline phosphatase, D day, Par Parnate

Wang et al. Stem Cell Research & Therapy (2017) 8:245 Page 7 of 13

preprogramming process. From days 9 to 11, the colonymass underwent massive death, and the surviving cellsgrew slowly to maturity. However, because early inductionusing the traditional episomal induction system (termed4F2L-6C), which includes Oct4, Sox2, Klf4, c-Myc, SV40-LT, and Lin28 [12], is a gradual process, the cells aggre-gated slowly and displayed little massive death (Fig. 5a,Additional file 9: Figure S6a, S6b); these results suggestthat the two induction systems differ regarding iPSC gen-eration. Therefore, we examined the safety of iPSCs gener-ated using the 6F/BM1-4C system.Evaluation of the application of iPSCs is based on safety.

Schlaeger et al. [36] analyzed chromosomal variation among

iPSCs induced using retrovirus, mRNA transfection,SeV, episomal, and lentivirus systems and observed largedifferences using these various methods. For example,the aneuploidy rate of iPSCs induced using the mRNAtransfection method was only 2.3%, whereas that ofiPSCs induced episomally was as high as 11.5%. How-ever, these methods employ different reprogramminggenes, and it is difficult to determine whether differ-ences in chromosomal variation reflect the genes orthe methods used for induction. In our study, we uti-lized an episomal vector to reprogram the same batchof hUCs isolated from the same donor using the 6F/BM1-4C and 4F2L-6C systems, and at least 60 iPSC

Table

1Indu

ctionefficiencyandcharacterizationof

iPSC

sindu

cedfro

mhU

Csusingthe6F/BM1-4C

system

Don

orAge

Gen

der

Celln

umbe

rDeath

rate

a(%)

Efficiency(%)

No.

Non

-integration

Karyotype

FACS

BSP

Teratomab

EBform

ation

Immun

ofluorescent

123

Female

3×10

653.8

0.0468

UC1iPSC1

+c

++

++

++

UC1iPSC2

+c

++

––

––

226

Male

2.8×10

680.37

0.0015

UC2iPSC1

+c

++

–+

++

UC2iPSC2

+c

++

––

–

327

Male

3×10

650

0.0741

UC3iPSC1

+c

+–

–+

+–

UC3iPSC2

+c

+–

+–

––

426

Female

3×10

671

0.00089

UC4iPSC1

+c

+–

––

––

UC4iPSC2

+c

+–

+e

––

–

524

Male

3×10

666.35

0.00095

UC5iPSC1

+c

+–

–+

––

UC5iPSC2

+c

+–

+e

––

–

630

Female

2.53

×10

636

0.00021

UC6iPSC1

+d

+–

––

––

UC6iPSC2

+c

+–

+–

––

729

Male

3×10

627.5

0.0429

UC7iPSC1

+c

+–

+e

––

–

UC7iPSC2

+c

+–

––

––

Atnu

cleo

fection,

thehU

Cpa

ssag

enu

mbe

rwas

2+iPSC

siden

tified,

–characterizationno

tiden

tified,

iPSC

indu

cedpluripoten

tstem

cell,hU

Chu

man

urine-de

rived

cell,FA

CSflu

orescence-activ

ated

cellsorting,

EBem

bryo

idbo

dy,H

Ehe

matoxylin

andeo

sin

a Takingsupe

rnatan

t(con

tainingno

nadh

eren

tcells)to

coun

tcells

onthe3rdda

yafterelectron

ictran

sformation,

theam

ount

ofcoun

tedcells

was

theam

ount

ofde

adcells.D

eath

rate

calculated

as:d

eath

rate

=am

ount

ofde

adcells/amou

ntof

cells

forelectrop

oration

bWeinjected

fivedifferen

tdo

nor-de

rived

iPSC

sin

theteratomaexpe

rimen

t.Each

NOD/SCID

mou

sewas

injected

with

acell.In

6–8weeks,three

NOD/SCID

miceform

edteratomas

andweredissectedan

dHEstaine

d.In

the8thweek,on

eNOD/SCID

miceha

dgran

ule-shap

edteratoma,an

dtheteratomawas

takenou

tan

dHEstaine

din

the12

thweek.One

mou

sedidno

tform

teratomaby

observation

c Exoge

nous

gene

sequ

ence

didno

tintegratein

thege

nome

dExog

enou

sge

nesequ

ence

integrated

inthege

nome

e Correspon

ding

prim

arycells

didno

tde

tect

thepluripoten

cyge

neprom

oter

DNAmethy

latio

ndu

eto

theexha

ustedcells

Wang et al. Stem Cell Research & Therapy (2017) 8:245 Page 8 of 13

a

1 2 3 4 5

6 7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 X Y

Oct4 Nanog

UC

1iP

SC

1U

C1

HN

4

d

Gut epithelium Cartilage Neural tissuef

c SSEA4/DAPIOCT4/DAPI

Tra-1-60/DAPI Tra-1-81/DAPI

Oct4Sox2

miR-302a

Nanog

Sox2Nanog

Oct4

miR-302a

g

b

103 1040 106105 103 1040 106105

103 1040 106105 103 1040 106105

99.1% 99.9%

96.9%90.5%

OCT4 SSEA4

Tra-1-60 Tra-1-81

UC1iPSC1Isotype

e

UC1iPSC1UC1iPSC1 EB

mesoderm

rela

tive

expr

essi

on le

vel

MSX1

Tbx5

GATA6

DESMIN

IGF2

0

925

5060

90 ****

*****

ectoderm

rela

tive

exp

ress

ion

leve

l

UC1iPSC1UC1iPSC1 EB

MAP2

SOX1

PAX6GFAP

0

1050

6095

100105 **

**

entoderm

rela

tive

expr

essi

on le

vel

UC1iPSC1UC1iPSC1 EB

AFP

FOXA2

SOX17FGF8

GATA4

Cytoke

ratin

8

Cytoke

ratin

180

730

4580

120

****

*

** ** *

** *

Fig. 4 Pluripotent characterization of iPSCs induced from UC1 cells using the 6F/BM1-4C system. a Karyotype analysis of iPSCs induced from UC1cells. b Flow cytometry assay for expression of the hESC markers OCT4, SSEA4, Tra-1-60, and Tra-1-81. c Immunofluorescence assay for expressionof hESC markers. d Bisulfite sequencing assay for the methylation status of the Oct4 and Nanog promoters in iPSCs. e In-vitro differentiation assayfor UC1 iPSCs, and EB morphology. f Hematoxylin and eosin staining of sections of teratomas generated from UC1 iPSCs. g Scatter plots comparingglobal gene expression patterns between HN4 hESCs and UC1 iPSCs and between UC1 cells and UC1 iPSCs. Highlighted are the pluripotency factorsOct4, Sox2, Nanog, and miR-302a. Error bars indicate mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 100 μm. EB embryoid body

Wang et al. Stem Cell Research & Therapy (2017) 8:245 Page 9 of 13

colonies (65 for 6F/BM1-4C and 64 for 4F2L-6C) were se-lected for karyotype analysis (the specific criteria are sum-marized in Methods). We observed a significantly lowerchromosomal abnormality rate for 6F/BM1-4C-iPSCs thanfor 4F2L-6C-iPSCs (P = 0.017, χ2 test; Fig. 5b, Table 2). Wealso determined the expression profiles of genes associatedwith genomic stability, such as Sirt1, p53, and CHK1, andfound significantly high Sirt1 expression in iPSCs inducedusing the 6F/BM1-4C system (Fig. 5c). These datashow that iPSCs induced using the 6F/BM1-4C sys-tem are safer than those induced using the traditionalepisomal induction method, which would be beneficialfor clinical applications.

DiscussionReflecting their high efficiency and controllable cost,episomal plasmid-carried reprogramming factors are themost widely used approach for obtaining non-integratediPSCs. Most episomal induction methods employ at leastone oncogene or tumorigenic molecule, such as c-Myc,SV40-LT, p53 short hairpin RNA (shRNA), and the p53small molecule inhibitor cyclin pifithrin-α [10, 12–17], andiPSCs induced using previous methods cannot be usedin clinical applications. Furthermore, induction methodsthat do not include tumorigenic factors are essential. Inthe present study, we constructed a low-risk 6F/BM1-4C reprogramming system, in which we eliminated the

c

TERT

0

1

2

3

rela

tive

expr

essi

on le

vel

6F/B

M1-

4C

4F2L

-6C

CHK1

0

1

2

2.5

rela

tive

expr

essi

on le

vel

6F/B

M1-

4C

4F2L

-6C

1.5

0.5

p53

0

1

2

2.5

rela

tive

expr

essi

on le

vel

6F/B

M1-

4C

4F2L

-6C

1.5

0.5

Sirt1

0

1

2

3

rela

tive

expr

essi

on le

vel

6F/B

M1-

4C

4F2L

-6C

*b

aD5 D13 D13D21

6F/B

M1-

4C

4F2L

-6C

D5 D21

0

5

10

15

20

25

Ab

no

rma

lka

ryo

typ

eR

ate

(%)

6F/B

M1-

4C

4F2L

-6C

*

Fig. 5 Safety comparison between 6F/BM1-4C and a traditional episomal induction system. a Changes in morphology in the iPSC generation processusing the 4F2L-6C and 6F/BM1-4C systems. b Abnormal karyotype rates of iPSCs generated using the 4F2L-6C (n= 64) and 6F/BM1-4C (n= 65) systems.*P< 0.05 (P= 0.017). c Quantitative real-time PCR assay for Sirt1, p53, TERT, CHK1 expression. *P< 0.05 (P= 0.034). Scale bars, 100 μm. D day

Wang et al. Stem Cell Research & Therapy (2017) 8:245 Page 10 of 13

tumorigenic factors used in traditional episomal repro-gramming systems, such as c-Myc, SV40-LT, and p53 in-hibitor, and included Oct4, Glis1, Klf4, Sox2, L-Myc, themiR-302 cluster and four compounds, and then treatedcells for no longer than 48 h to efficiently generate iPSCsfrom hUCs. This system also successfully converted hUCsfrom different sources into iPSCs and showed good repro-ducibility. Analyzing a large number of iPSCs by karyotypeanalysis, the 6F/BM1-4C-hiPSCs we generated exhibitedfewer chromosome abnormalities compared with trad-itional 4F2L-6C-hiPSCs. In addition, expression of Sirt1,the NAD-dependent deacetylase necessary for maintain-ing iPSC genomic stability [37], in 6F/BM1-4C-iPSCs washigh compared with iPSCs induced using the 4F2L-6Csystem, suggesting that 6F/BM1-4C-iPSC chromosomeinheritance is more stable. Moreover, the presentedmethod has a low cost, and the use of episomal plas-mids makes this system suitable for clinical non-integrated iPSC preparation.To obtain large-scale amounts of clinical-grade iPSCs, a

reprogramming method with good reproducibility, non-tumorigenic reprogramming factors, and cost-effectivenessis needed; xeno-free components and a medium for pri-mary somatic cell isolation to iPSC generation are also ne-cessary. Besides, although the 6F/BM1-4C reprogrammingsystem has relatively high reprogramming reproducibility,due to the heterogeneity of hUCs [18, 33, 34], it is difficultto accurately and separately perform multiplication culture

in vitro to obtain a variety of different types of cells thatmeet the required number of experiments; hUCs fromdifferent donors and different batches of cells alsoshow a wide range of induction efficiencies in the 6F/BM1-4C reprogramming system (Table 1). Therefore,except for a xeno-free induction reprogramming sys-tem, in the future the best reprogramming systemshould be screened for different types of hUCs or gen-eral suitability for a variety hUCs; in addition, the par-ticular type of hUCs that is more easily reprogrammedor the particular type of hUCs that is more suitable forthe 6F/BM1-4C system should be screened to find amore specific cultivation method for a particular typeof hUCs, so that we can obtain a high-efficiency repro-gramming system for screening high-quality clinical-gradeiPSCs from a large number of iPSCs. Furthermore, whenwe used the xeno-free hESC E8 medium [38] to inducehUC reprogramming based on episomal vectors, wefound it to be unsuitable after the addition of certaincompounds, with deformed cells all dying (data notshown). Xeno-free extracellular matrices such as Vitronec-tin exhibit poor maintenance of iPSC self-renewal capacity[38]. Conversely, Laminin521 maintains iPSC self-renewalcapacity, but it is extremely expensive [39] and thus isnot suitable for large-scale production of clinical-gradeiPSCs. Accordingly, the selection of appropriate cellculture materials remains essential for further clinicalapplications.

Table 2 Specific changes in cell karyotypes using the 4F2L-6Cand 6F/BM1-4C systems

No. Method Karyotype change(s)

7 4F2L-6C 88 < 4n>,XXXX,-X,-19,-18,-18[20]

13 4F2L-6C 46 < 2n>,XX,der(2)ins(2;?)(P25;?)[20],89 < 4n>,XXXX,-XX,-5[20]

14 4F2L-6C 87 < 4n>,XXXX,-XX,-15,-19,-19[20]

18 4F2L-6C 89 < 4n>,XXXX,-17,-17,-15?

19 4F2L-6C 46 < 2n>,XX,der(13)t(13;1)(p13;q11→ q44)[20]

22 4F2L-6C 45 < 2n>,XX,-17[20]

29 4F2L-6C 91 < 4n>,XXXX,der(15)t(15;22)(p13;p13→ q13),-5[20]

37 4F2L-6C 46 < 2n>,XX,+9[20]

40 4F2L-6C 45 < 2n>,XX,-7[20]/-17[20]/-21[20],47 < 2n>,XX,+15[20]

49 4F2L-6C 45 < 2n>,XX,-X[20], 92 <4n>,XXXX,der(17)ins(17;?)(p13;?)

54 4F2L-6C 92 < 4n>,XXXX,-X,+11[20]

57 4F2L-6C 45 < 2n>,XX,-9[20]/-17[20]

60 4F2L-6C 92 < 4n>,XXXX,-X,+22[20]

3 6F/BM1-4C 90 < 4n>,XXXX,-16,-16, 45 < 2n>,XX,-22[20]/-20[20]

15 6F/BM1-4C 47 < 2n>,XX,+9[20]

59 6F/BM1-4C 48 < 2n>,XX,+21,+22, 90 < 4n>,XXXX,-18,-20[20]/-21,-22[20]

61 6F/BM1-4C 47 < 2n>,XX,+X[20]/+9[20]

4F2L-6C episomal-induced system containing six reprogramming factors (Oct4,Sox2, Klf4, c-Myc, SV40-LT, and Lin28) and six compounds (PD, CHIR, NaB,Par, thi, and A83-01), 6F/BM1-4C episomal-induced system containing sixreprogramming factors (Oct4, Glis1, Klf4, Sox2, L-Myc, and miR-302cluster) andfour compounds (PD, CHIR, NaB, and Par), [20] 20 metaphases, <2n > abnormalfrequency≥ 3 metaphases, <4n > abnormal frequency≥ 15%Reference to International System for Human Cytogenetic Nomenclature 2009

Wang et al. Stem Cell Research & Therapy (2017) 8:245 Page 11 of 13

ConclusionWe developed a safe method based on an episomal vectorfor inducing iPSCs from hUCs. This method does not in-volve the use of tumorigenic factors, such as c-Myc, SV40-LT, and p53 inhibitor. Karyotype analysis revealed that thechromosomal variation that occurred during iPSC gener-ation in the present study was significantly low comparedwith the traditional method. Such low variability is criticalfor clinical applications of iPSCs.

Additional files

Additional file 1: Figure S1. showing expression of factors fromepisomal vectors. a pE3.1 plasmid construction process chart (upper).Schematic representation of seven constructed episomal vectors. pEF1αEF1α promoter, pCMV CMV promoter (below). b Quantitative real-timePCR assay for Oct4, Glis1, Klf4, Sox2, L-Myc, linc-RoR, miR-367, miR-302a,miR-302b, miR-302c, and miR-302d. c Western blot assay for Oct4, Glis1,Klf4, Sox2, and L-Myc carried on episomal vectors. GAPDH was used asthe loading control. (PDF 171 kb)

Additional file 2: Table S1. Presenting information for primers orfunctional fragments used in the present study. (XLSX 17 kb)

Additional file 3: Table S2. presenting plasmid combinations used forscreening low-risk factors. (XLSX 9 kb)

Additional file 4: Table S3. presenting the method of karyotype analysis.(XLSX 10 kb)

Additional file 5: Figure S2. showing use of hUC16 cells to screen forlow-risk factors for iPSC generation. a hUC16 morphology. b Strategy toscreen low-risk factor combinations using hUC16 cells. c AP staining foriPSC generation using different factor combinations. d Numbers of AP-positivecolonies. (PDF 137 kb)

Additional file 6: Figure S3. showing the effects of multiple compoundsin the 6F combination system. a Strategy to optimize six-factor combinationsusing A83-01. b AP staining for iPSCs induced using A83-01. c Number of AP-positive colonies induced using A83-01. P(B) = 0.002. d Strategy to optimizesix-factor combinations using Thiazovivin (thi). e AP staining of iPSCs inducedusing thi. f Number of AP-positive colonies induced using thi. P(D) = 0.000. gStrategy to optimize six-factor combinations using forskolin, PS48, and sc-79. hAP staining for iPSCs induced using forskolin, PS48, and sc-79. i Number ofAP-positive colonies induced using forskolin, PS48, and sc-79. j Strategy tooptimize six-factor combinations using DMOG and DZNEP. k AP staining foriPSCs induced using DMOG and DZNEP. l Number of AP-positive coloniesinduced using DMOG and DZNEP. P(C) = 0.041.m Strategy to optimize six-factor combination using Parnate in the early induction stage. n AP stainingfor iPSCs induced using Parnate in the early induction stage. Arrow indicatescell edge hemming. o Strategy to optimize six-factor combination treated withParnate for a short time. p AP staining for iPSCs induced using Parnate for ashort time. q Number of AP-positive colonies. P(C) = 0.04. Error bars indicatemean ± SD. *P< 0.05, **P< 0.01, ***P< 0.001. Scale bars, 100 μm. (PDF 227 kb)

Additional file 7: Figure S4. showing non-integrating analysis andkaryotype assays of iPSCs induced with the 6F/BM1-4C system. aNon-integrating analysis of genomic DNA in iPSCs. Representativelanes: 1, H2O; 2, pE3.1-OG- -KS and pE3.2-L-Myc- -hmiR-302 cluster; 3,UC5; 4, UC5, pE3.1-OG- -KS, and pE3.2-L-Myc- -hmiR-302 cluster; 5, UC5iPSC1;6, UC5iPSC2; 7, UC6; 8, UC6, pE3.1-OG- -KS, and pE3.2-L-Myc- -hmiR-302cluster; 9, UC6iPSC1; 10, UC6iPSC2; 11, UC7; 12, UC7, pE3.1-OG- -KS, andpE3.2-L-Myc- -hmiR-302 cluster; 13, UC7iPSC1; 14, UC7iPSC2. OriP in lane9 exhibited integration. b Karyotype analysis of iPSCs induced fromseveral hUCs. (PDF 340 kb)

Additional file 8: Figure S5. showing pluripotent characterization ofiPSCs induced using the 6F/BM1-4C system. a Flow cytometry for expressionprofiles of the hESC markers OCT4, SSEA4, Tra-1-60, and Tra-1-81. b Bisulfitesequencing assay for the methylation status of the Oct4 and Nanog promotersin iPSCs. Color codes indicate the proportion of methylation. y axis showsindividual CpGs analyzed. x axis shows different cells. c Immunofluorescenceassay for expression profiles of hESC markers. d Quantitative real-time PCRassay for expression profiles of marker genes of the three germ layers. eHematoxylin and eosin staining of sections of iPSC-generated teratomas. fScatter plots comparing global gene expression patterns between HN4 hESCsand UC1 iPSCs and between UC2 cells and UC2 iPSCs. Highlighted are thepluripotency factors Oct4, Sox2, Nanog, and miR-302a. Error bars indicatemean ± SD. *P< 0.05, **P< 0.01, ***P< 0.001. Scale bars, 100 μm. (PDF 375 kb)

Additional file 9: Figure S6. showing morphology changes duringiPSC generation using the 6F/BM1-4C system. a Morphology alteredduring iPSC generation using the 6F/BM1-4C system. b Schematic ofepisomal vectors used in the 4F2L-6C system. pEF1α EF1α promoter,pCMV CMV promoter. Scale bars, 100 μm. (PDF 158 kb)

AbbreviationsiPSC: Induced pluripotent stem cell; hUC: Human urine-derived cell;hESC: Human embryonic stem cell; MCS: Multiple cloning site; AP: Alkalinephosphatase; EB: Embryoid body; PD: PD0325901; CHIR: CHIR99021;NaB: Sodium butyrate; Par: Parnate; thi: Thiazovivin; 4C: PD, CHIR, NaB, andPar; 4F2L-6C: episomal-induced system containing six reprogramming factors(Oct4, Sox2, Klf4, c-Myc, SV40-LT, and Lin28) and six compounds (PD, CHIR,NaB, Par, thi, and A83-01); 6C: PD, CHIR, NaB, Par, thi, and A83-01; 6F/BM1-4C: episomal-induced system containing six reprogramming factors (Oct4,Glis1, Klf4, Sox2, L-Myc, and miR-302 cluster) and four compounds (PD, CHIR,

Wang et al. Stem Cell Research & Therapy (2017) 8:245 Page 12 of 13

NaB, and Par); OSKM: Oct4, Sox2, Klf4, and c-Myc; 6F: Oct4, Glis1, Klf4, Sox2, L-Myc, and the miR-302 cluster; 4F2L: Oct4, Sox2, Klf4, c-Myc, SV40-LT, and Lin28

AcknowledgementsThe authors thank American Journal Experts for English editing.

FundingNot applicable.

Availability of data and materialsSupporting data are available from the corresponding author uponreasonable request.

Authors' contributionsLLW, LBS, and JL conceived and designed the experiments. LLW, YHC, CYG,ZJZ, QL, JGY, JM, BW, WW, and XHY performed the experiments. LLW, YHC,CYG, QL, and JGY analyzed the data. BW, WW, and XHY contributed reagents/materials/analysis tools. LLW, ZJZ, and JL drafted the paper. All authors read andapproved the final manuscript.

Ethics approval and consent to participatehUCs were obtained from subjects who provided written informed consentfor the use of their cells for iPSC generation, and the experiments involvinghuman subjects and animal research were reviewed and approved by theInstitutional Review Board at Guangzhou Biocare Institute of Cancer(BioCareIRB201304a#).

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Publisher's NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.

Author details1Guangzhou Biocare Institute of Cancer, Building D, Guangzhou InternationalBusiness Incubator, No. 3, Juquan Road, Guangzhou Science Park,Guangzhou 510663, Guangdong, People’s Republic of China. 2TheGuangdong Key Lab for Shock and Microcirculation Research, Departmentsof Pathophysiology, Southern Medical University, Guangzhou 510515,People’s Republic of China. 3State Key Laboratory of Oncology in SouthernChina and Department of Experimental Research, Sun Yat-sen UniversityCancer Centre, Guangzhou 510060, People’s Republic of China. 4Departmentof Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, 74Zhongshan Road II, Yuexiu District, Guangzhou, Guangdong 510080, China.

Received: 22 March 2017 Revised: 21 September 2017Accepted: 16 October 2017

References1. Ando M, Nishimura T, Yamazaki S, Yamaguchi T, Kawana-Tachikawa A,

Hayama T, Nakauchi Y, Ando J, Ota Y, Takahashi S, Nishimura K, Ohtaka M,Nakanishi M, Miles JJ, Burrows SR, Brenner MK, Nakauchi H. A safeguardsystem for induced pluripotent stem cell-derived rejuvenated T cell therapy.Stem Cell Rep. 2015;5:597–608.

2. Vizcardo R, Masuda K, Yamada D, Ikawa T, Shimizu K, Fujii S, Koseki H,Kawamoto H. Regeneration of human tumor antigen-specific T cells fromiPSCs derived from mature CD8(+) T cells. Cell Stem Cell. 2013;12:31–6.

3. Srivastava D, DeWitt N. In vivo cellular reprogramming: the next generation.Cell. 2016;166:1386–96.

4. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S,Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. Inducedpluripotent stem cell lines derived from human somatic cells. Science.2007;318:1917–20.

5. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, YamanakaS. Induction of pluripotent stem cells from adult human fibroblasts bydefined factors. Cell. 2007;131:861–72.

6. Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, Ebina W, Mandal PK,Smith ZD, Meissner A, Daley GQ, Brack AS, Collins JJ, Cowan C, SchlaegerTM, Rossi DJ. Highly efficient reprogramming to pluripotency and directeddifferentiation of human cells with synthetic modified mRNA. Cell Stem Cell.2010;7:618–30.

7. Zhou H, Wu S, Joo JY, Zhu S, Han DW, Lin T, Trauger S, Bien G, Yao S, ZhuY, Siuzdak G, Scholer HR, Duan L, Ding S. Generation of induced pluripotentstem cells using recombinant proteins. Cell Stem Cell. 2009;4:381–4.

8. Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M. Efficient induction oftransgene-free human pluripotent stem cells using a vector based onSendai virus, an RNA virus that does not integrate into the host genome.Proc Jpn Acad Ser B Phys Biol Sci. 2009;85:348–62.

9. Woltjen K, Michael IP, Mohseni P, Desai R, Mileikovsky M, Hamalainen R,Cowling R, Wang W, Liu P, Gertsenstein M, Kaji K, Sung HK, Nagy A.piggyBac transposition reprograms fibroblasts to induced pluripotent stemcells. Nature. 2009;458:766–70.

10. Yu J, Hu K, Smuga-Otto K, et al. Human induced pluripotent stem cells freeof vector and transgene sequences. Science. 2009;324:797-801.

11. Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent inducedpluripotent stem cells. Nature. 2007;448:313–7.

12. Yu J, Chau KF, Vodyanik MA, Jiang J, Jiang Y. Efficient feeder-free episomalreprogramming with small molecules. PLoS One. 2011;6:e17557.

13. Valamehr B, Robinson M, Abujarour R, Rezner B, Vranceanu F, Le T, MedcalfA, Lee TT, Fitch M, Robbins D, Flynn P. Platform for induction andmaintenance of transgene-free hiPSCs resembling ground state pluripotentstem cells. Stem Cell Reports. 2014;2:366–81.

14. Xue Y, Cai X, Wang L, Liao B, Zhang H, Shan Y, Chen Q, Zhou T, Li X, Hou J,Chen S, Luo R, Qin D, Pei D, Pan G. Generating a non-integrating human inducedpluripotent stem cell bank from urine-derived cells. PLoS One. 2013;8:e70573.

15. Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S, Hong H,Nakagawa M, Tanabe K, Tezuka K, Shibata T, Kunisada T, Takahashi M,Takahashi J, Saji H, Yamanaka S. A more efficient method to generateintegration-free human iPS cells. Nat Methods. 2011;8:409–12.

16. Okita K, Yamakawa T, Matsumura Y, Sato Y, Amano N, Watanabe A, GoshimaN, Yamanaka S. An efficient nonviral method to generate integration-freehuman-induced pluripotent stem cells from cord blood and peripheralblood cells. Stem Cells. 2013;31:458–66.

17. Li D, Wang L, Hou J, Shen Q, Chen Q, Wang X, Du J, Cai X, Shan Y, Zhang T,Zhou T, Shi X, Li Y, Zhang H, Pan G. Optimized approaches for generationof integration-free iPSCs from human urine-derived cells with smallmolecules and autologous feeder. Stem Cell Rep. 2016;6:717–28.

18. Zhou T, Benda C, Duzinger S, Huang Y, Li X, Li Y, Guo X, Cao G, Chen S, HaoL, Chan YC, Ng KM, Ho JC, Wieser M, Wu J, Redl H, Tse HF, Grillari J, Grillari-Voglauer R, Pei D, Esteban MA. Generation of induced pluripotent stem cellsfrom urine. Clin J Am Soc Nephrol. 2011;22:1221–8.

19. Wang L, Wang L, Huang W, Su H, Xue Y, Su Z, Liao B, Wang H, Bao X, QinD, He J, Wu W, So KF, Pan G, Pei D. Generation of integration-free neuralprogenitor cells from cells in human urine. Nat Methods. 2013;10:84–9.

20. Cheng L, Hu W, Qiu B, Zhao J, Yu Y, Guan W, Wang M, Yang W, Pei G.Generation of neural progenitor cells by chemical cocktails and hypoxia.Cell Res. 2014;24:665–79.

21. Nakagawa M, Takizawa N, Narita M, Ichisaka T, Yamanaka S. Promotion ofdirect reprogramming by transformation-deficient Myc. Proc Natl Acad SciU S A. 2010;107:14152–7.

22. Maekawa M, Yamaguchi K, Nakamura T, Shibukawa R, Kodanaka I, Ichisaka T,Kawamura Y, Mochizuki H, Goshima N, Yamanaka S. Direct reprogrammingof somatic cells is promoted by maternal transcription factor Glis1. Nature.2011;474:225–9.

23. Lin SL, Chang DC, Chang-Lin S, Lin CH, Wu DT, Chen DT, Ying SY. Mir-302reprograms human skin cancer cells into a pluripotent ES-cell-like state.RNA. 2008;14:2115–24.

24. Lin SL, Chang DC, Lin CH, Ying SY, Leu D, Wu DT. Regulation of somatic cellreprogramming through inducible mir-302 expression. Nucleic Acids Res.2011;39:1054–65.

25. Lin SL, Chang DC, Ying SY, Leu D, Wu DT. MicroRNA miR-302 inhibitsthe tumorigenecity of human pluripotent stem cells by coordinatesuppression of the CDK2 and CDK4/6 cell cycle pathways. Cancer Res.2010;70:9473–82.

26. Yan X, Lv Y, Ma J, et al. The development of extrahepatic portacaval shuntdevice based on magnetic compression technique through theinterventional procedure. Chin J Med Instrum. 2013;37:421-22, 36.

Wang et al. Stem Cell Research & Therapy (2017) 8:245 Page 13 of 13

27. Loewer S, Cabili MN, Guttman M, Loh YH, Thomas K, Park IH, Garber M,Curran M, Onder T, Agarwal S, Manos PD, Datta S, Lander ES, Schlaeger TM,Daley GQ, Rinn JL. Large intergenic non-coding RNA-RoR modulatesreprogramming of human induced pluripotent stem cells. Nat Genet.2010;42:1113–7.

28. Meisner LF, Johnson JA. Protocols for cytogenetic studies of humanembryonic stem cells. Methods. 2008;45:133–41.

29. Xu Y, Zhu X, Hahm HS, Wei W, Hao E, Hayek A, Ding S. Revealing a coresignaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules. Proc Natl Acad Sci U S A. 2010;107:8129–34.

30. Li W, Zhou H, Abujarour R, Zhu S, Young Joo J, Lin T, Hao E, Scholer HR,Hayek A, Ding S. Generation of human-induced pluripotent stem cells inthe absence of exogenous Sox2. Stem Cells. 2009;27:2992–3000.

31. Zhu S, Li W, Zhou H, Wei W, Ambasudhan R, Lin T, Kim J, Zhang K, Ding S.Reprogramming of human primary somatic cells by OCT4 and chemicalcompounds. Cell Stem Cell. 2010;7:651–5.

32. Hou P, Li Y, Zhang X, Liu C, Guan J, Li H, Zhao T, Ye J, Yang W, Liu K, Ge J,Xu J, Zhang Q, Zhao Y, Deng H. Pluripotent stem cells induced from mousesomatic cells by small-molecule compounds. Science. 2013;341:651–4.

33. Rahmoune H, Thompson PW, Ward JM, Smith CD, Hong G, Brown J.Glucose transporters in human renal proximal tubular cells isolated fromthe urine of patients with non-insulin-dependent diabetes. Diabetes. 2005;54:3427–34.

34. Dorrenhaus A, Muller JI, Golka K, Jedrusik P, Schulze H, Follmann W. Culturesof exfoliated epithelial cells from different locations of the human urinarytract and the renal tubular system. Arch Toxicol. 2000;74:618–26.

35. Zhou T, Benda C, Dunzinger S, Huang Y, Ho JC, Yang J, Wang Y, Zhang Y,Zhuang Q, Li Y, Bao X, Tse HF, Grillari J, Grillari-Voglauer R, Pei D, EstebanMA. Generation of human induced pluripotent stem cells from urinesamples. Nat Protoc. 2012;7:2080–9.

36. Schlaeger TM, Daheron L, Brickler TR, Entwisle S, Chan K, Cianci A, DeVine A,Ettenger A, Fitzgerald K, Godfrey M, Gupta D, McPherson J, Malwadkar P,Gupta M, Bell B, Doi A, Jung N, Li X, Lynes MS, Brookes E, Cherry AB,Demirbas D, Tsankov AM, Zon LI, Rubin LL, Feinberg AP, Meissner A, CowanCA, Daley GQ. A comparison of non-integrating reprogramming methods.Nat Biotechnol. 2015;33:58–63.

37. De Bonis ML, Ortega S, Blasco MA. SIRT1 is necessary for proficient telomereelongation and genomic stability of induced pluripotent stem cells. StemCell Rep. 2014;2:690–706.

38. Beers J, Gulbranson DR, George N, Siniscalchi LI, Jones J, Thomson JA, ChenG. Passaging and colony expansion of human pluripotent stem cells byenzyme-free dissociation in chemically defined culture conditions. NatProtoc. 2012;7:2029–40.

39. Rodin S, Antonsson L, Hovatta O, Tryggvason K. Monolayer culturing andcloning of human pluripotent stem cells on laminin-521-based matricesunder xeno-free and chemically defined conditions. Nat Protoc. 2014;9:2354–68.

• We accept pre-submission inquiries

• Our selector tool helps you to find the most relevant journal

• We provide round the clock customer support

• Convenient online submission

• Thorough peer review

• Inclusion in PubMed and all major indexing services

• Maximum visibility for your research

Submit your manuscript atwww.biomedcentral.com/submit

Submit your next manuscript to BioMed Central and we will help you at every step: